• Cryo-EM structure of Vibrio cholerae EIIC transporter in substrate-free inward-facing state at 3.68 Å resolution reveals a larger substrate-binding pocket poised for glucose release.
• A unique intraprotomer disulfide bond (C240-C254) stabilizes the scaffold-transport domain interface, potentially regulating transporter dynamics.
• Comparative analysis with E. coli EIIC structures highlights conformational changes essential for alternating access and substrate release.
• EIIC transporter emerges as a promising antimicrobial target; disrupting sugar transport could impair Vibrio cholerae growth and virulence, offering a novel therapeutic strategy against cholera.